The 5' noncoding region of grapevine chrome mosaic nepovirus RNA-2 triggers a necrotic response on three Nicotiana spp.

The 5' noncoding region (NCR) of grapevine chrome mosaic nepovirus (GCMV) was cloned in a viral vector derived from potato virus X (PVX). The recombinant virus obtained was inoculated to Nicotiana benthamiana, N. clevelandii, and N. tabacum plants. Infected plants developed necrotic symptoms in place of the vein clearing and mosaic typically observed after inoculation with PVX. Northern (RNA) blot analysis showed that the replication of PVX was not specifically altered by the presence of the GCMV 5' NCR. Inoculation of recombinant PVX harboring deleted forms of the GCMV 5' NCR showed that the three stem-loop structures at the 3' end of the 5' NCR (nucleotides 153 to 206) are dispensable for the induction of necrosis. Further deletion analysis indicated that neither the 5'-most 70 nucleotides of the 5' NCR nor the downstream region (nucleotides 71 to 217) alone is able to induce the necrotic symptoms. In the presence of both the sequence encoding the GCMV coat protein and the GCMV 3' NCR, the GCMV 5' NCR failed to induce necrosis in the PVX background. The mechanisms by which the expression of the 5' NCR might modify PVX symptoms are discussed.

Construction of a chimeric viral gene expressing plum pox virus coat protein.

The capsid-encoding gene of plum pox virus (PPV) was fused with the leader sequence of the coat protein mRNA (cp) of tobacco mosaic virus by a novel mutagenesis technique which involves reverse transcription of minus-strand RNA [synthesized by in vitro transcription of a double-stranded (ds) cDNA clone], using an ad hoc synthetic oligodeoxynucleotide as primer. The resulting cDNA was rendered ds and cloned into the plasmid, pBluescribe M13+. Transcription of this chimeric construction produced RNA molecules of 1250 nucleotides in length, which were used as messengers in the in vitro protein-synthesizing systems. The major product of this transcript consists of a 36-kDa polypeptide and was identified as the PPV coat protein (CP) by molecular weight estimation and by immunoprecipitation with a polyclonal antiserum to PPV. Transfer of this cDNA via Agrobacterium tumefaciens into plants was successfully performed. Transgenic Nicotiana plants producing the PPV CP were subsequently obtained.

Cloning full-length cDNA of grapevine chrome mosaic nepovirus.

Full-length cDNA copies of the genomic RNAs of grapevine chrome mosaic virus were obtained and cloned in Escherichia coli by a one-step procedure. The cloning protocol included size selections by agarose-gel electrophoresis of both the single-stranded and the double-stranded full-length cDNAs. First-strand cDNA synthesis was primed with oligodeoxythymidine while second-strand synthesis was primed with specific synthetic oligodeoxynucleotides, allowing cloning of the 3' poly(A) and of the last 5' nucleotides of the viral RNA template. For the 7.2-kb and 4.4-kb viral RNAs, up to 20% and 80%, respectively, of the clones were found to be full-length. Even for large templates, this procedure allows fast and efficient cloning of full-length cDNAs.

Comparison of the complete nucleotide sequences of two isolates of lettuce mosaic virus differing in their biological properties.

The complete nucleotide sequences of the genomic RNAs of the 0 and E isolates of lettuce mosaic potyvirus (LMV) have been determined. These two isolates differ by their behavior towards two lettuce resistance genes and by their seed transmission properties. LMV-0 is unable to induce disease in lettuce carrying either one of the mol1 and mol2 recessive resistance genes, whereas LMV-E is able to induce disease in the same plants. The genomes of these two isolates are 10080 nucleotides (nt) in length, excluding the poly(A) tract, and encode polyproteins of 3255 amino acids (aa). The open reading frame is flanked by a 5' non-coding region of 103 nt and a 3' non-coding region of 212 nucleotides. Ten proteins were predicted. The P3 protein, with 377 aa, is the longest potyviral P3 protein characterized to date while the P1 protein, with 437 aa, is among the longest P1 proteins reported. Sequence comparisons between the two isolates demonstrated only limited sequence difference. The overall nucleotide and amino acid sequence identities between LMV-0 and LMV-E are 94 and 97% respectively. The greatest variability occurs in the P1 and in the variable N-terminal region of the coat protein, while the NIa protease domain, the NIb protein, the C-terminus of the helper component protease and the 3' non-coding region are extensively conserved. While this sequence analysis does not allow direct identification of determinants involved in the resistance breaking or in the seed transmissibility properties, these data are a first step towards the characterization of these determinants.

Production and use of monoclonal antibodies for the detection of apple chlorotic leaf spot virus.

Stable hybridoma cell lines secreting antibodies specific for the apple chlorotic leaf spot virus (CLSV) were produced by fusing spleen cells of a Biozzi mouse immunized with CLSV P863 strain, with the non-secretory P3 X63 Ag8.653 myeloma cell line. Two hybridoma clones producing monoclonal antibodies of the IgG1 subclass were obtained. These monoclonal antibodies were used for virus detection by enzyme-linked immunosorbent assay (ELISA). In contrast to polyclonal antisera to CLSV, which always contain some antibodies to host components, monoclonal antibodies are highly specific for the virus. It was thus possible to develop a detection assay which is more sensitive and specific than the assays using polyclonal antibodies. Using monoclonal antibodies, it was possible to detect less than 0.1 ng/ml of purified virus. In addition, these two monoclonal antibodies recognize 17 strains or isolates maintained in our laboratory and representing most of the known CLSV strains.

Detection of Chrysanthemum stunt viroid (CSV) using nick translated probes in a dot-blot hybridization assay.

We developed a dot-blot hydridization assay for the detection of Chrysanthemum stunt viroid (CSV) in Chrysanthemum plant samples. The probe, a recombinant plasmid containing a full-length monomeric cDNA copy of CSV, is labelled with (32P) by nick-translation. The influence of the hybridization conditions, of the sample denaturation technique and of the plant sap components on the final sensitivity has been studied. The optimized system, involving a formaldehyde denaturation step, allows the detection of as little as 5 pg of purified viroid. Under these conditions, 100 pg of pure viroid diluted in plant sap, or infected plant extract diluted 1:25 in healthy extract can be detected, showing the potential of this method for indexing of Chrysanthemum for CSV infection.

A polymerase chain reaction assay adapted to plum pox potyvirus detection.

A sensitive, polyvalent assay based on the polymerase chain reaction (PCR) was developed for plum pox potyvirus (PPV) detection. This technique was adapted for a single tube, the chemical denaturation and reverse transcription of the viral RNA followed by the PCR reaction yielding a 243-base-pair product. As few as 10 fg of purified viral RNA, corresponding to approximately 2000 viral particles, were detected in plant extracts. All PPV isolates tested were amplified, and the amplified fragments were analysed by restriction endonuclease digestion. An RsaI restriction site polymorphism in the amplified fragments allowed the discrimination of two groups of isolates. In a field indexing trial, the PCR assay proved to be more sensitive than molecular hybridization using 32P-labelled RNA probes for PPV detection.

A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection.

A highly sensitive assay, based on polymerase chain reaction amplification of cDNA synthesized from the viral RNA of antibody-captured viral particles, has been developed for plum pox potyvirus (PPV) detection. The reaction, called immunocapture/PCR (IC/PCR), yields a specific 243-bp product. The immunocapture step, by allowing the use of large sample volumes and by the viral particle prepurification it achieves, dramatically increases the sensitivity of the assay. As few as 8000 target viral particles per ml of plant extract could be detected by IC/PCR. When compared to direct PCR (Wetzel et al., 1991), molecular hybridization using 32P-labeled cRNA probes and ELISA, this result corresponds to a 250-fold, 625-fold and 5000-fold increased sensitivity, respectively. The high sensitivity of IC/PCR was confirmed during an indexing trial with field samples collected from naturally infected trees. This very powerful technique should have wide ranging applications for the detection of a number of other viruses and pathogens for which specific antisera and sequence data are available.

Dot hybridization detection of plum pox virus using 32P-labeled RNA probes representing non-structural viral protein genes.

A cDNA library covering the complete genome of plum pox virus strain D (PPV D) has been obtained, and an endonuclease restriction map derived from it. This map was superposed on the PPV genomic organisation map, established for a nonaphid transmissible strain of PPV (Maiss et al., 1989). This allowed us to select seven probes, corresponding to different regions on the PPV genome. These probes were tested in a dot-blot hybridization assay for the detection of PPV. Probes of various lengths (0.25 to 1.5 kb) were tested and those measuring at least 0.8 kb (4 of the 7 probes selected) proved to be the most sensitive. The detection limit was of about 5 pg of purified virus per assay. Probes representing non-structural viral protein genes were equally sensitive in detecting both serotypes D and M of PPV. The previously described probe pBPPV1 (Varveri et al., 1988), covering the coat protein gene of strain D, was less sensitive, when compared to the above probes, in detecting heterologous strains of PPV. The polyvalence of probes transcribed from non-structural viral protein genes was confirmed by screening isolates of PPV, collected in infected orchards in several Mediterranean countries.

Biological and molecular variability of lettuce mosaic virus isolates.

ABSTRACT Lettuce mosaic potyvirus (LMV) causes severe disease of commercial lettuce crops. LMV isolates show wide biological variability, particularly in their ability to overcome the resistance genes described in Lactuca sativa. For a better understanding of the molecular interaction between lettuce and LMV, biological and molecular characterization of a collection of 10 LMV isolates known to differ in virulence or aggressiveness was performed. The ability of these isolates to overcome the resistance genes was reevaluated under standardized conditions. To study the molecular variability of LMV, an immunocapture-reverse transcription-poly-merase chain reaction technique, coupled with direct sequencing, was used to obtain nucleotide sequence data from three short regions of the LMV genome. Clustering analysis was performed and compared to the biological properties of the 10 isolates. Three groups of LMV isolates were discriminated based on the molecular data. These groups appear to correlate with the geographic origin of the isolates rather than with their pathogenicity. Sequence comparison with California isolates clearly showed that the California isolates are related to the western European isolates, raising the possibility of past exchanges of LMV between western Europe and California.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

Transgenic tobaccos transformed with a gene encoding a truncated form of the coat protein of tomato black ring nepovirus are resistant to viral infection.

Tomato black ring virus (TBRV) belongs to the nepoviruses, an important genus of phytoviruses characterized by isometric particles and by their transmission by longidorid nematodes. As for all other nepoviruses, the coat protein (CP) of TBRV is encoded by the 3' terminal part of the viral RNA2 (positions 2801-4334). A hybrid gene driving the expression of a truncated form of the TBRV CP was constructed. It contains a frameshift deletion at position T4065 so that in the encoded protein the last 90 amino acids of the wild-type CP are replaced by 52 amino acids encoded by a different reading frame of the viral RNA. This hybrid gene was introduced into the genome of Nicotiana tabacum cv 'Xanthi' plants. When compared to control plants, progeny of some transformants expressing the mutated CP gene (CPm+ plants) showed resistance against TBRV infection. This resistance is characterized by a delay in the appearance of symptoms, a reduction in the number of infected plants and a reduction in virus accumulation.

Resistance of Transgenic Prunus domestica to Plum Pox Virus Infection.

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.

High resistance and control of biological risks in transgenic plants expressing modified plum pox virus coat protein.

Transgenic plums transformed with the plum pox virus coat protein (PPV CP) gene displayed a resistance to the sharka disease (Ravelonandro et al., 1997). However, the expression of PPV CP in transgenic plants may lead to complementation of deficient characteristic of an incoming potyvirus. Indeed, an aphid-intransmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) could be transmitted when encapsidated by the engineered PPV CP (Lecoq et al., 1993). To control such a risk, new PPV CP constructs were designed and introduced into Nicotiana benthamiana genome. In the first construct, the DAG amino acid triplet involved in the potyvirus aphid-transmission was deleted. The second construct encoded a truncated PPV CP lacking its first 140 amino acids. In the last construct, the nucleotides encoding the charged amino-acids R220, Q221 and D264 localized in the core of the PPV CP were removed. A bacterial expression system was developed to show that these deletions prevent the assembly of the PPV CP subunits. For each construct, several transgenic lines were produced and first challenged with several strains of PPV. Two phenotypes of resistance were observed: recovery and immunity. Their biochemical characterization showed that the resistance was RNA-mediated and therefore can be classified as homology-dependent (Jacquet et al., 1998a). Resistant lines producing high level of wild type or modified PPV CP were then inoculated with ZYMV-NAT to perform an aphid-transmission assay. Results of these experiments demonstrated that the use of modified forms of PPV CP genes in transgenic plants provide a good way to control the biological risks associated with heteroencapsidation (Jacquet et al., 1998b).

Characterization of phenotype resistance to plum pox of transgenic plums expressing plum pox virus capsid gene.

Resistance to plum pox virus (PPV) infection can be obtained in transgenic plants that express the virus capsid gene. An Agrobacterium-mediated transformation was used to introduce the PPV capsid gene into Prunus domestica plants. Over 11 regenerated plants (clones) were observed for the development of the disease symptoms and analysed for the presence of PPV by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) through 4 dormancy cycles. The level of protection against PPV was determined in the transformed plants, non-transformed plants, and a control transgenic plant "transformed" with the plasmid vector alone. One clone, C-5, appeared fully protected, while PT-6 and C-4 clones accumulated a low concentration of virus and the rest of the clones was entirely susceptible. Little is known about the mechanisms of resistance to virus infection in transgenic woody plants. To investigate this aspect, comparative studies based on the characteristics of resistant and susceptible clones have been started. A question, whether the phenotype resistance of clone C-5 is similar to that observed in transgenic herbaceous plants or not, has been addressed. Recent progress in this investigation is presented.

Use of monoclonal antibodies for the identification of different antigenic domains in apple chlorotic leaf spot virus.

Thirteen monoclonal antibodies (Mabs) specific for apple chlorotic leaf spot virus (ACLSV), produced by the somatic cell hybridization technique, were used to investigate the antigenic structure of the virus. Epitope specificity studies showed that these Mabs defined in ACLSV particles seven independent antigenic domains, representing at least eight distinct epitopes. One of them was present only in virions and not in dissociated subunits. It appeared that the interaction between a Mab and the virus could, in some cases, induce conformational changes in the viral particles which enhanced the binding of others. Twenty nine virus isolates differing in geographical origin, primary hosts and symptomatology were tested with these monoclonal antibodies by ELISA. With the exception of two Mabs which did not react with three cherry isolates, and one Mab which did not react with one plum isolate, all of them recognized all ACLSV isolates tested.

Transfer of the 3' non-translated region of grapevine chrome mosaic virus RNA-1 by recombination to tomato black ring virus RNA-2 in pseudorecombinant isolates.

In grapevine chrome mosaic and tomato black ring viruses (GCMV and TBRV), as in many other nepoviruses, the 3' non-translated regions (3'NTR) are identical between the two genomic RNAs. We have investigated the structure of the 3'NTR of two recombinant isolates which contain GCMV RNA-1 and TBRV RNA-2. In these isolates, the 3'NTR of RNA-1 was transferred to RNA-2, thus restoring the 3' identity. The transfer occurred within three passages, and probably contributes to the spread of randomly appearing mutations from one genomic RNA to the other. The site of recombination is near the 3' end of the open reading frame.

Nucleotide sequence of the 3' ends of the double-stranded RNAs of grapevine chrome mosaic nepovirus.

Attempts were made to label the termini of dsRNAs corresponding to the two genomic RNAs of grapevine chrome mosaic nepovirus (GCMV). It was not possible to label the 5' ends of the dsRNAs with [gamma-32P]ATP, which suggests that a genome-linked protein blocks their 5' ends. Both dsRNA species were labelled at their 3' ends with pCp. The 3'-terminal sequences were determined by 'wandering spot' or by partial enzymic cleavage analysis. One strand (presumably positive) ended in a poly(A) 30 to 50 nucleotides long whereas the other (presumably negative) ended in 3'-ACCUUUUAAAAAG (RNA1) or 3'-ACCUUUUAAUAAAG (RNA2). The sequences resemble closely those complementary to the 5' ends of the RNAs of tomato black ring virus (strain S), which is distantly related to GCMV.